Gene targeting demonstrates that inducible nitric oxide synthase is not essential for resistance to oral candidiasis in mice, or for killing of Candida albicans by macrophages in vitro.

INTRODUCTION: Oral candidiasis is caused by opportunistic infections with the yeast Candida albicans. Previous studies have demonstrated important roles for innate immunity and T helper type 1-mediated inflammatory reactions in recovery from infection, with macrophages and neutrophils as key effector cells. Both effector cell types use the inducible isoform of nitric oxide synthase (iNOS) to generate candidacidal molecules, but it is not clear whether nitric oxide (NO) is an absolute requirement for candidacidal effector activity. METHODS: In this study we directly investigated the role of iNOS-derived NO in resistance to murine experimental oral candidiasis, using iNOS knockout mice. RESULTS: Knockout mice were no more susceptible to oral candidiasis than wild-type controls. Bone marrow-derived macrophages from the knockout mice killed C. albicans yeasts efficiently in vitro, and were still able to produce nitrites in an iNOS-independent manner, albeit less efficiently than wild-type controls. There were no significant differences in local mucosal production of interleukins 6, 12, 17A, or 23, interferon-gamma, or transforming growth factor-beta 24 h after oral challenge with C. albicans. CONCLUSION: These data suggest that iNOS-derived NO is not required for resistance to oral candidiasis in vivo, and that bone marrow-derived macrophages may have iNOS-independent means of generating reactive nitrogen species.

Distinct roles for interleukin-12p40 and tumour necrosis factor in resistance to oral candidiasis defined by gene-targeting.

Cell-mediated immunity is important for anti-Candida host defence in mucosal tissues. In this study we used cytokine-specific gene knockout mice to investigate the requirement for T helper type 1 (Th1) and Th2 cytokines in recovery from oral candidiasis. Knockout mice used in this study included interleukin-4 (IL-4), IL-10, IL-12p40, interferon-gamma (IFN-gamma), and tumour necrosis factor (TNF). The mice were challenged either orally or systemically with Candida albicans yeasts, and levels of colonization were determined. IL-12p40 knockout mice developed chronic oropharyngeal candidiasis, but were not more susceptible to systemic challenge. On the other hand, TNF knockout mice displayed increased susceptibility to both oral and systemic challenge, but only in the acute stages of infection. TNF apparently has a protective effect in the acute stages of both oral and systemic candidiasis, whereas IL-12p40 is essential for recovery from oral but not systemic candidiasis. The role of IL-12p40, and its relation to T-cell-mediated responses remain to be determined.

Isolates of Candida albicans that differ in virulence for mice elicit strain-specific antibody-mediated protective responses.

Three distinct isolates of Candida albicans were used to establish systemic and oral infections in inbred mice that are genetically resistant or susceptible to tissue damage. Patterns of infection differed significantly between both yeasts and mouse strains. Systemic infection conferred significant protection against re-challenge with the homologous, but not the heterologous yeast; however, the protective effect was more evident in the tissue-susceptible CBA/CaH mice than in the resistant BALB/c strain. In contrast, oral infection induced protection against both homologous and heterologous oral challenge, although this was significant only in the CBA/CaH mice. CBA/CaH mice produced antibodies of both IgG1 and IgG2a subclasses, whereas BALB/c mice produced predominantly IgG1. Western blotting demonstrated considerable differences between epitopes recognised by serum antibodies from mice of both strains after immunisation with each of the three yeasts. Thus, different strains of yeast show considerable specificity in antibody responses elicited by either systemic or oral infection.

Active and passive immunization against oral Candida albicans infection in a murine model.

BACKGROUND/AIMS: Clinical and laboratory studies are consistent with a major role for cell-mediated immunity in recovery from oral infection with Candida albicans, but the role of humoral immunity remains controversial. The purpose of this study was to establish the relative contributions of cellular and humoral immunity to protection against oral candidiasis in a murine model, and to determine whether host responses could be enhanced by different immunization strategies. RESULTS: Active oral immunization was protective in BALB/c and CBA/CaH mice, reducing both fungal burden and duration of infection after secondary challenge, whereas systemic immunization failed to protect against subsequent oral challenge. Candida-specific IgM was the predominant antibody detected in serum following both primary and secondary oral challenge; however, Candida-specific salivary IgA was not detectable. Immunization by passive transfer of either lymphocytes or immune serum did not confer any significant protection against oral infection in either susceptible or resistant mouse strain. CONCLUSION: The data demonstrate a possible role for mucosa-associated immunity following active immunization by the oral route, most likely exerted by local T lymphocytes resident in the oral mucosa, but there was no evidence to support a role for humoral immunity in protection against oral candidiasis.

Modulation of the antibody response by Porphyromonas gingivalis and Fusobacterium nucleatum in a mouse model.

Successive immunization of mice with Fusobacterium nucleatum and Porphyromonas gingivalis has been shown to modulate the specific serum IgG responses to these organisms. The aim of this study was to investigate these antibody responses further by examining the IgG subclasses induced as well as the opsonizing properties of the specific antibodies. Serum samples from BALB/c mice immunized with F. nucleatum (gp1-F), P. gingivalis (gp2-P), P. gingivalis followed by F. nucleatum (gp3-PF) F. nucleatum followed by P. gingivalis (gp4-FP) or saline alone (gp5-S) were examined for specific IgG1 (Th2) and IgG2a (Th1) antibody levels using an ELISA and the opsonizing properties measured using a neutrophil chemiluminescence assay. While IgG1 and IgG2a subclasses were induced in all immunized groups, there was a tendency towards an IgG1 response in mice immunized with P. gingivalis alone, while immunization with F. nucleatum followed by P. gingivalis induced significantly higher anti-P. gingivalis IgG2a levels than IgG1. The maximum light output due to neutrophil phagocytosis of P. gingivalis occurred at 10 min using nonopsonized bacteria. Chemiluminescence was reduced using serum-opsonized P. gingivalis and, in particular, sera from P. gingivalis-immunized mice (gp2-P), with maximum responses occurring at 40 min. In contrast, phagocytosis of immune serum-opsonized F. nucleatum demonstrated peak light output at 10 min, while that of F. nucleatum opsonized with sera from saline injected mice (gp5-S) and control nonopsonized bacteria showed peak responses at 40 min. The lowest phagocytic response occurred using gp4-FP serum-opsonized F. nucleatum. In conclusion, the results of the present study have demonstrated a systemic Th1/Th2 response in mice immunized with P. gingivalis and/or F. nucleatum with a trend towards a Th2 response in P. gingivalis-immunized mice and a significantly increased anti-P. gingivalis IgG2a (Th1) response in mice immunized with F. nucleatum prior to P. gingivalis. Further, the inhibition of neutrophil phagocytosis of immune serum-opsonized P. gingivalis was modulated by the presence of anti-F. nucleatum antibodies, while anti-P. gingivalis antibodies induced an inhibitory effect on the phagocytic response to F. nucleatum.

Differences in mouse strain influence leukocyte and immunoglobulin phenotype response to Porphyromonas gingivalis.

This study examined the nature of the infiltrating cells in Porphyromonas gingivalis-induced lesions and immunoglobulins in the serum samples of BALB/c (H-2d), C57BL6 (H-2b), DBA/2J (H-2d) and CBA/CaH (H-2k) mice. Mice were immunized intraperitoneally with P. gingivalis outer membrane antigens or sham-immunized with phosphate-buffered saline followed by subcutaneous challenge with live organisms 1 week after the final immunization. The resulting skin abscesses were excised 7 days later, cryostat sections cut and an immunoperoxidase method used to detect the presence of CD4+ and CD8+ T-cell subsets, CD14+ macrophages and CD19+ B cells. Peroxidase positive neutrophils and IgG1- and IgG2a-producing plasma cells were also identified. Anti P. gingivalis IgG1 and IgG2a subclass antibodies were determined in serum obtained by cardiac puncture. Very few CD8+ T cells and CD19+ B cells were found in any of the lesions. The percentages of CD4+ cells, CD14+ cells and neutrophils were similar in lesions of immunized BALB/c and C57BL6 mice, with a trend towards a higher percentage of CD14+ cells in sham-immunized mice. The percentage of CD14+ cells was higher than that of CD4+ cells in immunized compared with sham-immunized DBA/2J mice. The percentages of CD4+ and CD14+ cells predominated in immunized CBA/CaH mice and CD4+ cells in sham-immunized CBA/CaH mice. The percentage of neutrophils in immunized CBA/CaH mice was significantly lower than that of CD14+ cells and CD4+ cells in sham-immunized mice. IgG1+ plasma cells were more dominant than IgG2a+ cells in immunized BALB/c, C57BL6 and DBA/2J mice, whereas IgG2a+ plasma cells were more obvious in sham-immunized mice. IgG2a+ plasma cells were predominant in immunized and sham-immunized CBA/CaH mice. In the serum, specific anti-P. gingivalis IgG2a antibody levels (Th1 response) were higher than IgG1 levels (Th2 response) in sham-immunized CBA/CaH and DBA/2J mice. In immunized BALB/c mice, IgG2a levels were lower than IgG1 levels, while IgG2a levels were higher in immunized C57BL6 mice. In conclusion, this study has shown differences in the proportion of infiltrating leukocytes and in the subclasses of immunoglobulin produced locally and systemically in response to P. gingivalis in different strains of mice, suggesting a degree of genetic control over the response to P. gingivalis.

Cytokines in the oral mucosa of mice infected with Candida albicans.

Oropharyngeal candidiasis is associated with defects in cell-mediated immunity, and is commonly seen in immunocompromised patients. We have previously shown that T-cell-deficient BALB/c nude (nu/nu) mice are extremely susceptible to oropharyngeal candidiasis, and that recovery from a chronic infection is dependent on CD4 T lymphocytes. In this study we describe the local tissue cytokine profile in lymphocyte-reconstituted immunodeficient mice and their euthymic counterparts. Mice were infected orally with 108 cells of the yeast Candida albicans, and oral tissues sampled on days 0, 4, 8, and 14. Nude mice were reconstituted with 3 x 107 naïve lymphocytes following oral inoculation. Interleukin (IL)-6, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha were identified in the oral tissues of infected euthymic mice recovering from oral infection, as well as naïve controls. TNF-alpha was identified in nude oral tissue on days 4 and 8, but only after lymphocyte reconstitution. No IL-2, IL-4 or IL-10 was detected in either euthymic or athymic mice at any time-point throughout the experiment. This study confirms the functional activity of T lymphocytes in reconstituted nude mice, and suggests that TNF-alpha may be an important mediator in the recovery from oropharyngeal candidiasis.

Primary role for CD4(+) T lymphocytes in recovery from oropharyngeal candidiasis.

Oropharyngeal candidiasis is associated with defects in cell-mediated immunity and is commonly seen in human immunodeficiency virus positive individuals and AIDS patients. A model for oral candidiasis in T-cell-deficient BALB/c and CBA/CaH nu/nu mice was established. After inoculation with 10(8) Candida albicans yeasts, these mice displayed increased levels of oral colonization compared to euthymic control mice and developed a chronic oropharyngeal infection. Histopathological examination of nu/nu oral tissues revealed extensive hyphae penetrating the epithelium, with polymorphonuclear leukocyte microabscess formation. Adoptive transfer of either naive or immune lymphocytes into immunodeficient mice resulted in the recovery of these animals from the oral infection. Reconstitution of immunodeficient mice with naive CD4(+) but not CD8(+) T cells significantly decreased oral colonization compared to controls. Interleukin-12 and gamma interferon were detected in the draining lymph nodes of immunodeficient mice following reconstitution with naive lymphocytes. This study demonstrates the direct requirement for T lymphocytes in recovery from oral candidiasis and suggests that this is associated with the production of cytokines by CD4(+) T helper cells.

Irradiation-induced oral candidiasis in an experimental murine model.

The aim of this experiment was to establish a mouse model of irradiation-induced oral candidiasis and to explore the cellular populations and mechanisms by which the infection is cleared from the oral mucosa. BALB/c mice received irradiation to the head and neck equivalent to 800 Rad using a Cobalt 60 gamma source. Both irradiated and non-irradiated mice were infected orally with 1 x 10(8) Candida albicans yeasts. Compared with untreated controls, irradiated animals developed a more severe infection of longer duration, with hyphae penetrating the oral mucosa. Monoclonal antibody depletion of CD4+ but not CD8+ T cells from the systemic circulation prolonged the infection in irradiated mice, but not in controls. Supernatants of submandibular and superficial cervical lymph node cultures from irradiated animals demonstrated significantly higher titers of interleukin-12, but similar levels of interferon-gamma compared with controls. Screening for cytokine production by an RNase protection assay detected only macrophage migration inhibition factor in irradiated and non-irradiated oral tissues from day 8 onwards. The results of this study demonstrate a requirement for CD4+ T cells in the recovery from oral candidiasis induced by head and neck irradiation in mice, and are consistent with a role for Th1-type cytokines in host resistance.

T cells augment monocyte and neutrophil function in host resistance against oropharyngeal candidiasis.

The purpose of this study was to identify the cell populations involved in recovery from oral infections with Candida albicans. Monoclonal antibodies specific for CD4+ cells, CD8+ cells, and polymorphonuclear leukocytes were used to deplete BALB/c and CBA/CaH mice of the relevant cell populations in systemic circulation. Monocytes were inactivated with the cytotoxic chemical carrageenan. Mice were infected with 10(8) C. albicans yeast cells and monitored for 21 days. Systemic depletion of CD4+ and CD8+ T lymphocytes alone did not increase the severity of oral infection compared to that of controls. Oral colonization persisted in animals treated with head and neck irradiation and depleted of CD4+ T cells, whereas infections in animals that received head and neck irradiation alone or irradiation and anti-CD8 antibody cleared the infection in a comparable fashion. The depletion of polymorphonuclear cells and the cytotoxic inactivation of mononuclear phagocytes significantly increased the severity of oral infection in both BALB/c and CBA/CaH mice. High levels of interleukin 12 (IL-12) and gamma interferon (IFN-gamma) were produced by lymphocytes from the draining lymph nodes of recovering animals, whereas IL-6, tumor necrosis factor alpha, and IFN-gamma were detected in the oral mucosae of both naïve and infected mice. The results indicate that recovery from oropharyngeal candidiasis in this model is dependent on CD4+-T-cell augmentation of monocyte and neutrophil functions exerted by Th1-type cytokines such as IL-12 and IFN-gamma.

Mechanical testing of a single rod versus a double rod in a long-segment animal model.

This study involved the mechanical testing of single-rod segmental hook fixation and double-rod segmental hook fixation in a long-segment animal model. The goals were first to compare the flexibility of a single-rod scoliosis construct with that of a double-rod construct when tested in torsion, and second, to determine the effect of not using instrumentation with every vertebral segment for the single rod. Another study found that the single-rod construct was as stiff in torsion as the standard double-rod construct in a model of 10 vertebral segments. The amount of neutral zone (NZ) rotation was tested in five calf spines using an MTS (Material Testing System) machine. Five constructs were tested and included 1) a single rod with hooks at every level except the apex; 2) a single rod with two fewer hooks; 3) a single rod with four fewer hooks; 4) a double-rod construct; and 5) no instrumentation. The amount of NZ rotation between vertebral segments was measured over 12, 10, 8, 6, 4, and 2 vertebral segments. An analysis of variance with all constructs showed that the instrumented spines had significantly less movement than did the uninstrumented spine. Statistical comparison using analysis of variance of constructs (constructs 1 to 4) showed that over 12 vertebral segments (T4-L3), all single-rod constructs (constructs 1 to 3) allowed more NZ rotation than did the standard double-rod construct. This testing indicated that over 12 vertebral segments the single rod allowed more NZ rotation than a double-rod construct.

Nitric oxide-enhanced resistance to oral candidiasis.

A murine model of oral candidiasis was used to show that nitric oxide (NO) is involved in host resistance to infection with Candida albicans in infection-'resistant' BALB/c and infection-'prone' DBA/2 mice. Following infection, increased NO production was detected in saliva. Postinfection samples of saliva inhibited the growth of yeast in vitro. Treatment with NG-monomethyl-L-arginine (MMLA), an inhibitor of NO synthesis, led to reduced NO production, which correlated with an increase in C. albicans growth. Reduction in NO production following MMLA treatment correlated with an abrogation of interleukin-4 (IL-4), but not interferon-gamma (IFN-gamma), mRNA gene expression in regional lymph node cells. Down-regulation of IL-4 production was accompanied with an increase in IFN-gamma production in infection-'prone' DBA/2 mice. There was a functional relationship between IL-4 and NO production in that mice treated with anti-IL-4 monoclonal antibody showed a marked inhibition of NO production in saliva and in culture of cervical lymph node cells stimulated with C. albicans antigen. The results support previous conclusions that IL-4 is associated with resistance to oral candidiasis and suggest that NO is involved in controlling colonization of the oral mucosal surface with C. albicans.

Cellular and cytokine correlates of mucosal protection in murine model of oral candidiasis.

Host protection against Candida albicans infection in a model of oral candidiasis involving infection-prone [DBA/2 (H-2(d))] and less infection-prone [BALB/c (H-2(d))] mouse strains was analyzed in terms of antibody and cellular responses, and in terms of cytokine patterns from regional lymph node cells. There was a selective expansion of gamma/delta(+) T-cell receptor cells, which correlated with the patterns of colonization in both mouse strains, with higher numbers of gamma/delta T cells detected in BALB/c mice. Antigen-induced T-cell proliferation was significantly higher in BALB/c mice than in DBA/2 mice. Higher levels of serum immunoglobulin G (IgG) and salivary IgA antibodies were detected in BALB/c mice than in DBA/2 mice, but only after the infection was cleared. The cervical lymph node cells from infected mice were assessed for interleukin-4 (IL-4), IL-12, and gamma interferon (IFN-gamma) mRNA gene expression by reverse transcription-PCR and protein production in the culture supernatants following restimulation in vitro. In BALB/c mice, an early increase in levels of IL-4, IFN-gamma, and IL-12 correlated with rapid elimination of C. albicans. In DBA/2 mice, where resolution of infection was delayed, IL-4 message expression was delayed and the IL-4 secretion level was lower. Neutralization of IL-4 by multiple injections of an anti-IL-4 monoclonal antibody in BALB/c mice resulted in increased carriage rate and delayed clearance of the yeasts. Collectively, the data suggest that the T-cell response to C. albicans in the regional lymph nodes which correlates best with rapid oral clearance of C. albicans is a balanced Th0 cytokine response involving early secretion of both IFN-gamma and IL-4.

Complement-mediated regulation of Trichomonas vaginalis infection in mice.

Trichomonas vaginalis is a flagellated protozoan which causes trichomoniasis, a sexually transmitted disease of the human genitourinary tract. The importance of the alternative complement pathway in host defence against T. vaginalis was investigated in vitro. Kinetic studies utilising immunofixation following electrophoresis showed that both a strongly and weakly virulent strain of T. vaginalis activated murine serum C3. In vivo studies with congenic-resistant, C5-deficient, B10.D2/OSn- and C5-sufficient, B10.D2/nSn mice showed that the presence of C5 is a significant factor in the innate host resistance to primary infection with a strongly virulent, but not a weakly virulent trichomonad strain.

In vivo evaluation of intramedullary interlocking nail fixation of transverse femoral osteotomies in foals.

OBJECTIVE: To evaluate an intramedullary interlocking nail for stabilization of transverse femoral osteotomies in foals. STUDY DESIGN: A transverse osteotomy and restabilization with an intramedullary interlocking nail was performed on the right femur in three foals and the left femur in three foals. ANIMALS: Six foals weighing 149 to 207 kg. METHODS: The femur was destabilized with a transverse middiaphyseal osteotomy and repaired with a 0.5-in (12.7 mm) interlocking nail. The implanted femurs were radiographed monthly until completion of the study 6 months after surgery. At the completion of the study, all foals were observed for evidence of lameness, gluteal thickness was determined by ultrasonographic measurement, and a necropsy was performed. RESULTS: Healing was satisfactory in all foals. Five of the six had osseous bridging of the osteotomy apparent radiographically by 3 to 4 months. The sixth foal had postoperative infection but was healed radiographically in 5 months. There was a mean decrease in gluteal muscle thickness of 6.6 mm (P = .04) in the operated limb of the five foals that healed without complication. Two foals were lame at the completion of the project; one foal with varus deformities of the contralateral limb was mechanically lame, and another was grade 2/5 lame on the operated limb. On necropsy, there was circumferential enlargement of the diaphysis of all operated limbs with the majority of the callus at the cranial and medial aspects of the cortex. All nails were solid within the medullary cavity. CONCLUSIONS: The intramedullary interlocking nail provided adequate stabilization for repair of the transverse osteotomy. CLINICAL RELEVANCE: Further investigation is warranted before use for stabilization of spontaneously occurring fracture configurations.

Type 1 and type 2 cytokines: from basic science to fungal infections.

At the present time, it is clear that Th1 responses afford protection against the fungi; however, the development, maintenance and function of the protective immune responses are complex mechanisms and are influenced by multiple factors. The route of infection has been shown to affect initial cytokine production and, consequently, the induction of protective Th1 responses. The ability of different isolates of the same fungal agent to induce and sustain a protective response has also been emphasized. Protective immune responses have been shown to vary in genetically different mouse strains after infection. In addition, these protective responses, such as cellular influx and cytokine production, also vary within the same animal depending on the tissue infected. The functional dominance of certain cytokines over others in influencing development and maintenance of protective responses has been discussed. Certain cytokines may act differently in hosts lacking important components of their innate or immune repertoire. It is evident from these presentations that a more comprehensive understanding of the protective mechanisms against different fungal agents is emerging. However, there is still much to learn before cytokine modulatory therapy can be used effectively without risk in the human host.

A gene (Carg1) that regulates tissue resistance to Candida albicans maps to chromosome 14 of the mouse.

Tissue susceptibility and resistance to infection with the yeast Candida albicans is genetically regulated. Analysis of the strain distribution pattern of the C. albicans resistance gene (Carg1) and additional gene and DNA segment markers in the AKXL recombinant inbred (RI) set showed that 13/15 RI strains were concordant for Carg1, Tcra and Rib1. Therefore, Carg1 is probably located within a 17 cM segment of chromosome 14, within approximately 4 cM of the other two genes.